WO2005120484A1 - Régulateur pour les fonctions physiologiques de la ghrelin - Google Patents

Régulateur pour les fonctions physiologiques de la ghrelin Download PDF

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WO2005120484A1
WO2005120484A1 PCT/JP2004/015413 JP2004015413W WO2005120484A1 WO 2005120484 A1 WO2005120484 A1 WO 2005120484A1 JP 2004015413 W JP2004015413 W JP 2004015413W WO 2005120484 A1 WO2005120484 A1 WO 2005120484A1
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darelin
ghrelin
glyceryl
acid
acyl
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Japanese (ja)
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Masayasu Kojima
Yoshihiro Nishi
Kenji Kangawa
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Kurume University
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Definitions

  • the present invention relates to regulators of the physiological function of darelin and their use in connection with the manufacture of pharmaceutical compositions or foods.
  • Darrelin is an endogenous ligand (peptide) of a receptor (GHS-R) that binds to growth hormone secretagogue (GHS), a synthetic non-natural substance that promotes growth hormone secretion. Hormone), which was the first substance discovered by the group of the present inventors [(1) and WO01 / 007475]. Initially, darelin was purified from the stomach of rats, but it has been demonstrated that it is also expressed in brain, lung, kidney, spleen, small and large intestine (2-7).
  • Ghrelin has also been isolated from cDNA from vertebrates other than rats, such as humans, mice, pigs, chickens, eel, sea lions, pomas, higgies, potatoes, -jimas or dogs, or has been estimated to have cDNA power ( JP-A-2004-2378).
  • Darrelin has an activity to increase intracellular calcium ion concentration and a potent growth hormone secretion-promoting activity (1, 8-10), and stimulates appetite, induces obesity (11--14), and improves cardiac function (15-17), has various activities such as promoting gastric acid secretion (18).
  • a potent growth hormone secretion-promoting activity (1, 8-10)
  • stimulates appetite induces obesity (11--14), and improves cardiac function (15-17)
  • has various activities such as promoting gastric acid secretion (18).
  • modulation of that function is important not only for subjects suffering from diseases related to Darrelin but also for healthy subjects.
  • Darrelin which has been identified so far, is a group of peptides having about 30 or less amino acid residues, and has a structural feature that the amino acid at position 3 is substituted with an acyl group.
  • human darelin consists of 28 amino acids, and the serine side chain at position 3 is acylated with a fatty acid (n-octanoic acid).
  • the amino acid 3 at the 3-position is essential for the expression of physiological activities such as the increase in intracellular calcium ion concentration of dallelin and the promotion of growth factor secretion (1).
  • the amino acid at position 3 of the darelin molecule is usually serine (hereinafter, referred to as “Ser 3 ” or “ser (3)”). No.
  • the acyl group used for modification of the amino acid at position 3, which is essential for the biological activity of darelin, is mainly a medium-chain / long-chain fatty acid residue.
  • Humans, pigs, sea lions, sheep, dogs, rats, mice, etc., mammals,-birds, such as birds, eel,-fish, such as jimas, terravia, catfish, etc., and amphibians, such as power frogs, are n-otatanyl. [1), (19), and
  • acyl modification examples include an n-decanoyl (C10: 0) modification (e.g., Shiga frog, Patent Document 2) and an n-decenoyl (C10: l) modification (20-22).
  • n-butanoyl (C4) e.g, Puma
  • hexanoyl (C6) e.g., hexanoyl (C6)
  • dodecanoyl (C12) are also known (Japanese Patent Application Laid-Open No. 2004-2378).
  • Ghrelin-acyl modification is the first example of lipid modification of peptide hormones, and serylhydrido-xyl-group acylation has never been reported as a modification of mammalian proteins.
  • the power of the presence of asilyi darelin and non-asiyi darelin in the living body The putative enzyme that catalyzes the transfer of the acyl group to the amino acid residue at position 3 of darelin is probably a novel acyltransferase, which regulates darelin production. Seems important. However, such enzymes have not yet been discovered.
  • a substance that modulates rouge at the 3-position amino acid in vivo in a living body functions as a "regulator” or “modulator” of the physiological function (activity) of darrellin) and various physiological physiological functions of ghrelin. It is expected to be useful for enhancing or suppressing the activity.
  • Such regulators can be used in the manufacture of a pharmaceutical composition for treating or preventing various physiological disorders related to the physiological activity of dallelin. Specific examples include pharmaceutical compositions for treating diseases caused by deficiency, decrease, or excess of growth hormone. In addition, it can be used for animals with anorexia and malnutrition, and animals exhibiting symptoms related to treatment such as health disorders related to excessive appetite and obesity. Or fattening of livestock is also useful for promoting growth and reducing fat
  • infusions or liquid diets used during treatment usually contain only minimal nutrients and are not necessarily effective in positively improving physical functioning. Therefore, in order to improve body functions quickly and effectively, there is a demand for the development of infusions and liquid foods with higher functions. Therefore, modulators of the physiological activity of dallelin are considered to be extremely useful for various uses, such as the functional foods described above, infusions, liquid foods, and livestock feeds.
  • Another object of the present invention is to provide a method for increasing or decreasing the concentration of modified darelin.
  • the present inventors have studied various synthetic-type acyl-modified darelin peptides, and as a result, have found that the effect of the biological activity of darelin can be modified by changing the acyl molecule ( twenty three).
  • the present inventors have found that ingested (exogenous) fatty acids are directly used in vivo for acylation of glycerin 3-position amino acids (e.g., Ser (3)).
  • the present inventors have found that such compounds are useful for controlling the physiological function of darelin, and have completed the present invention.
  • the present invention provides
  • 2.Darelin's physiological functions are an increase in intracellular calcium ion concentration, a promotion of growth hormone secretion, a promotion of feeding, a regulation related to fat accumulation, a cardiac function improvement or a gastric acid secretion stimulation.
  • the regulator described in 1 The regulator described in 1,
  • a method comprising administering to a subject in need of treatment for a disorder related to the physiological function of dallelin, the regulator according to 1 or the pharmaceutical composition according to 3 in a therapeutically effective amount. How to treat disorders related to the physiological function of
  • the regulator of the present invention affects the acylation of the amino acid at position 3 of endogenous darelin, and increases or decreases the ratio of modified darelin to various physiological disorders related to the physiological activity of darelin. Is effective in treating or preventing illness, in particular, treating diseases caused by growth hormone deficiency, reduction, or excess, anorexia, and malnutrition. Further, the regulator of the present invention is also useful, for example, for improving the growth of livestock. Furthermore, it may contribute to elucidation of the mechanism of the modification of the peptide hormone darelin to acil, particularly to the characterization of the putative darelin ser Q-acyl transferase.
  • N-RIA is very specific for acyl-modified darrelin and the main form of acylated ghrelin is n-otatanyl ghrelin
  • concentration of acyl-modified darellin measured by N-RIA is mainly n-otalinyl.
  • C represents the ratio of acyl-modified darelin Z total darelin.
  • the data represent the mean SD of darelin concentration in gastric extracts (from lmg wet weight). Statistical significance is indicated by an asterisk. *, p ⁇ 0.01; **, p ⁇ 0.001 vs. control.
  • FIG. 2 Standard stomach of mice fed a diet mixed with glyceryl trihexanoate (C6), glyceryl trioctanoate (C8), glyceryl tridecanoate (C10) or glyceryl tripalmitate (C16).
  • A represents the concentration of acyl-modified darelin measured by darelin N-RIA.
  • B represents total darelin concentration measured by ghrelin C-RIA.
  • arrows indicate the elution positions of desyl-type darelin (I) and n-otatanyl darrelin (II).
  • peaks a, d, h, and k correspond to the peaks of desacyl ghrelin
  • peaks b, f, i, and 1 correspond to the peaks of n-otatanyl dallelin.
  • Peaks g, j, and m corresponded to the peak of n-decenoyl (C10: l) ghrelin
  • peak n corresponded to the peak of n-decanoyl (C10: 0) dalelin.
  • FIG. 4 shows the time-dependent change in the stomach darrellin concentration of mice fed glyceryl trioctanoate.
  • A represents the content of the acyl-modified darelin measured by darelin N-RIA.
  • FIG. 5 shows Northern blot analysis for testing gastric darelin mRNA expression after ingestion of a glyceryl trioctanoate-containing diet. Each lane contains 2 ⁇ g of total RNA. The lower panel shows 28S and 18S ribosomal RNA internal controls.
  • FIG. 6 shows an HPLC profile of a gastric extract derived from a mouse fed with glyceryl triheptanoate.
  • Gastric extracts of mice treated with glyceryl triheptanoate were fractionated by HPLC (upper panel).
  • Darrelin concentration in each fraction (0.2 mg equivalent of gastric tissue) was monitored by C-RIA (middle panel) and N-RIA (lower panel).
  • C-RIA methicillin-associated ANC
  • N-RIA lower panel
  • darelin immunoreactivity was separated by C-RIA into three major peaks (middle panel, peaks a, b and c) and by N-RIA two major peaks (peaks d and e). ). Peaks b and d were only observed after ingestion of glyceryl triheptanoate.
  • FIG. 7 shows the final purification of n-heptanoyldarellin.
  • Stomach strength of mice receiving glyceryl triheptanoate also purified the darelin peptide.
  • the sample from which the anti-rat ghrelin immobility column was also eluted was subjected to HPLC. Peak a was only observed in samples from mice treated with glyceryl triheptanoate. HPLC retention time and
  • peak b was! /, Corresponding to n-otatanyldarellin. Arrows indicate elution positions of n-hexanoyl (1), n-otatanyl ( ⁇ ) and n-decanoyl (III) ghrelin, respectively.
  • FIG. 8 A is a matrix-assisted laser of dallelin-like peptide purified from peak a in Figure 7 4 shows the results of desorption ionization time-of-flight mass spectrometry. Mass ranges from 3131.0 to 3477.0 (m / z). From the average 100 mass spectra obtained in the positive ion mode (average [M + H] +: 3301.9), the molecular weight of peak a peptide was calculated to be 3300.9.
  • B Structure of n-heptanoyl (C 7: 0) ghrelin. The calculated molecular weight of n-heptanoyldarellin is 3300.86.
  • FIG. 9 shows the molecular form of plasma darelin peptide derived from mice fed a glyceryl triheptanoate mixed diet.
  • Plasma samples from control mice (A) and daliseryl triheptanoate-treated mice (B) fed a standard diet were fractionated by HPLC and ghrelin immunoreactivity was measured by C-RIA.
  • Arrows indicate elution positions of desacyl-type darelin (I) and n-otatanyl darrelin ( ⁇ ).
  • the plasma dalelin immunoreactivity was represented by a bar graph.
  • peaks b and e correspond to deotathanildarellin
  • peaks c and g correspond to n-otatanildarellin.
  • the newly appearing peak f showed the same retention time as n-heptanoyldarellin observed in mouse stomach after glyceryl tryptanoate treatment.
  • “Darelin” is a peptide hormone of about 30 amino acid residues that binds to the endogenous growth hormone secretagogue (GHS) receptor GHS-R and has the activity of increasing intracellular calcium ion concentration and stimulating growth hormone secretion. It is. Darrelin is widely distributed in vertebrates and has been identified in mammals, birds, fish, and amphibians. Thus, the present invention encompasses darellin from any source.
  • GHS growth hormone secretagogue
  • Preferred sources of ghrelin include humans, pigs, sea lions, horses, wedges, egrets, rats, mice, dogs, -birds, puppies, -jimas, edible powers, and other livestock, poultry, pet fish, etc. is there.
  • Several darelins from these animals have already been isolated and their amino acid sequences are known. For example, see JP-A-2004-2378. .
  • (acyl) -modified darelin refers to the amino acid residue at position 3 (eg, serine) of a darelin molecule having a specific amino acid sequence exemplified in SEQ ID NOS: 13 to 13. Modified with a group Peptide, also referred to simply as “acyl ghrelin”.
  • acylation means that the side chain hydroxyl group of the amino acid at position 3 is replaced with an acyl group, preferably a fatty acid residue.
  • unmodified darelin means a peptide in which the 3-position amino acid is not acylated, and is also simply referred to as “deacyldarelin”.
  • the term "regulator" of the physiological function of ghrelin means a substance that enhances or weakens the physiological function of darellin when administered to a living body expressing RHS-R using ghrelin as a ligand.
  • Examples of the substance that enhances the physiological function of darrellin include a fatty acid having an activating effect and having an acyl group at which dallin is physiologically active when the amino acid at the 3-position of darrellin is acylated.
  • a substance that weakens the physiological activity of dallelin it does not affect or rather reduces the physiological activity of dallelin. Can be exemplified.
  • mice In the case of mice as described in Examples below, the intake of medium chain fatty acid (MCFA) or medium chain triacylglycerol (MCT) is determined by the total ghrelin (acyl ghrelin and deacil ghrelin) concentration. The production of acyl-modified ghrelin was increased without altering ghrelin.
  • MCFA medium chain fatty acid
  • MCT medium chain triacylglycerol
  • dalelin peptides modified with n-butyryl or n-palmitoyl groups were undetectable after ingestion of the corresponding short (SCFA) or long (LCFA) chains.
  • n-heptanoyl ghrelin (a non-natural form of darelin) was produced in the stomach of mice after ingestion of glyceryl n-heptanoate or triheptanoate.
  • mice in which darelin is acylated by medium-chain fatty acids medium-chain fatty acids (n-hexanoic acid, n-octanoic acid and n-decanoic acid) or medium-chain triglycerides (glyceryl trihexanoate) are used.
  • Glyceryl trioctanoate and glyceryl tridecanoate are taken up by darellin modified by an acyl group having a carbon chain of the corresponding length (i.e., n-hexanoyldarellin, n-otatanyldarellin). And n-decanoyldarellin) in the stomach.
  • the ingested fatty acids and triglycerides are used as a lipid source for the modification of darelin to acyl, and affect the concentration of the acyl-modified darelin, and thus function as a regulator of the physiological function of darelin.
  • fatty acids that increase the physiological function of darrellin when bound to the amino acid at position 3 of darrellin are ⁇ positive regulators, '' while fatty acids that do not affect or inhibit the physiological function of darrellin are: It can function as a "negative regulator".
  • the present invention will be mainly described with reference to Darrelin in which the 3-position amino acid is serine as an example.
  • the present invention is also applied to a Darrelin homologue in which the 3-position amino acid is threonine, and the same effect is obtained. What can be obtained can be easily understood by those skilled in the art.
  • a "regulator of the physiological function of darrellin” at least one of darrellin by having a fatty acid moiety capable of forming an ester with the hydroxyl group of the 3-position amino acid (e.g., Ser (3)) of the darrellin molecule. Substances that regulate one function.
  • Fatty acids that can be used as the active ingredient of the regulator of the present invention include saturated or unsaturated fatty acids having 2 to 35 carbon atoms. Specific examples include butanoic acid (C4), hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), dodecanoic acid (C12), tetradecanoic acid (C14), and hexadecanoic acid having an even number of carbon atoms.
  • C16 octadecanoic acid
  • C18 pentanoic acid with an odd number of carbon atoms
  • C5 heptanoic acid
  • C9 nonanoic acid
  • C17 heptadecanoic acid
  • their monoenes or Polyene fatty acids and the like C16, octadecanoic acid (C18), pentanoic acid with an odd number of carbon atoms (C5), heptanoic acid (C7), nonanoic acid (C9)
  • pentadecanoic acid C15
  • heptadecanoic acid C17
  • fatty acid having 418 carbon atoms More preferably, it is a fatty acid having 418 carbon atoms, more preferably a fatty acid having 6-16 carbon atoms. Power is not limited to these.
  • the fatty acids that can be used vary depending on the target animal, but usually have a carbon number of 412, preferably 8—. 10, most preferably between 6-10.
  • octanoic acid preferably, power prillic acid
  • decanoic acid preferably, power pric acid
  • dodecanoic acid preferably, lauric acid
  • the fatty acids that can be used are those that differ depending on the target animal. Usually, they are other than the fatty acids exemplified as the positive regulators described above. . That is, those having carbon atoms other than 411, more preferably other than 6-10 can be exemplified.
  • Derivatives of fatty acids are mentioned. Such derivatives may also be converted into salts or esters as appropriate for the purpose of improving solubility, gastrointestinal absorption, taste and odor.
  • a method for producing such a derivative is well known in the field of manufacturing industry for pharmaceuticals, foods, feeds, and the like, and those skilled in the art can produce an appropriate derivative according to the purpose.
  • esters with mono- or polyalcohols which are usually used for similar purposes.
  • glycerin is a preferred alcohol.
  • glycosides they may be mono-, di- or triglycerides or mixtures thereof, with triglycerides being most preferred.
  • the fatty acid or a derivative thereof as an active ingredient of the regulator of the present invention can be obtained according to a method known to those skilled in the field of organic chemistry or a commercially available power source.
  • Physiological functions of darelin that can be controlled by the regulator of the present invention include all physiological functions of isildarin, for example, the effect of increasing intracellular calcium ion concentration.
  • a growth hormone secretion promoting action a feeding promoting action, a regulation action related to fat accumulation, a cardiac function improving action or a gastric acid secretion stimulating action.
  • it is involved in, but not limited to, growth hormone release, appetite stimulation, obesity induction, cardiac function improvement, and gastric acid secretion.
  • the regulator of the present invention enhances the physiological function of darellin, the effect of the regulator is similar to that of darellin or an analog thereof. That is, the regulator may have effects such as promotion of growth hormone secretion, stimulation of appetite, induction of obesity, improvement of cardiac function, stimulation of secretion of gastric acid, and the like.
  • Such a regulator is given to mammals, birds, fish, amphibians, and the like, for example, humans, pigs, pacific horses, magpies, sheep, egrets, rats, mice, dogs, chicks, penguins, rainbow trouts, and the like. The above effects are exhibited.
  • drugs for eating disorders drugs for promoting growth hormone secretion, drugs for heart disease, drugs for gastric functional diseases, drugs for protecting intestinal mucosa or agents for preventing small intestinal mucosal damage during parenteral nutrition, drugs for treating osteoporosis, It is useful as an agent for reducing cachexia due to chronic diseases and as a therapeutic agent for pulmonary dysfunction. In particular, it is useful for preventing or treating osteoporosis, anorexia, heart disease, rheumatism and inflammatory bowel disease in humans, and promoting recovery after surgery.
  • a pharmaceutical composition comprising a regulator of darellin physiological function.
  • the fatty acid or derivative thereof of the present invention can be used as it is because it functions as a regulator of the physiological function of darellin of the present invention itself.For ease of handling or application, fatty acid or its derivative is used. It is preferred to formulate in a suitable form, including liquid and solid forms according to methods known in the art. Examples include solutions and suspensions in aqueous or non-aqueous media (diluents), powders, granules or tablets with physiologically acceptable or pharmaceutically acceptable carriers. Such a pharmaceutical composition can enhance or suppress the function of darelin in various animal species described in the section “Physiological function of darelin,” for example, and exhibit the therapeutic effects described in the same section. it can.
  • the regulator of the physiological function of darelin of the present invention is formulated into a pharmaceutical composition, it is formulated by a method known per se using excipients, solvents, carriers, preservatives and the like known to those skilled in the art. Is done.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intradermal, subcutaneous, intravenous injection, drip, etc.) by a method known in the medical or veterinary field.
  • the dosage of the regulator of the present invention varies depending on various factors (the selected fatty acid or its derivative, the administration route, and the subject to be treated, including the disorder to be treated, age, weight, condition, etc.), and is usually Determined by a physician.
  • Based on fatty acids a force of between O.OOlmg-1000 mg, preferably between O.OOlmg-100 mg, more preferably O.Olmg-10 mg. Such a range is not limiting.
  • the dose is appropriately determined by a veterinarian or the like according to the subject.
  • the regulator of the present invention can be used as a functional food for promoting or suppressing appetite, relieving obesity, improving malnutrition, and the like. In particular, it can be used to control the health of mammals by controlling body weight, etc., and also to promote animal growth and reduce fat in meat. Thus, the regulator of the present invention is also useful in livestock raising, poultry farming, fish farming, and the like.
  • a functional food can be produced according to a method known in the art, for example, food, feed, edible oil, and soft drink. What is necessary is just to make it contain in water, infusion liquid, liquid food, etc. Alternatively, the regulator may be mixed with the normal diet before use.
  • the content of the regulator of the present invention in the functional food can be appropriately determined by those skilled in the art based on the dosage described in the section of the pharmaceutical composition.
  • the treatment of disorders related to the physiological function of dallelin using the regulator of the present invention is well known in the art by giving the regulator itself or a pharmaceutical composition containing the same to humans or non-human animals. Can be carried out according to the method described in
  • GHS Growth hormone secretagogue
  • GHS-R Growth hormone secretagogue receptor
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • N-RIA N-terminal fragment of n-otatanyl ghrelin [1-11]
  • Radioimnoassay C-RIA ghrelin Radioimnoassay of C-terminal fragment of [13-28]
  • MCFA Medium chain fatty acids
  • the darelin-specific RIA was performed according to the method described in the literature (2 above).
  • the two polyclonal antibodies to fragments and C-terminal (Gln 13 -Arg 28) fragment (Glyi-Lys 11 having at the Qn- Otatanoirui spoon Ser 3) Rattogureri emissions of N-terminal was induced in Usagi.
  • the RIA incubation mixture is mixed with standard ghrelin or unknown sample 1001. Diluted with RIA buffer (50 mM sodium phosphate buffer (pH 7.4), 0.5% BSA, 0.5% Triton-X100, 80 mM NaCl, 25 mM EDTA-2Na and 0.05% NaN) containing 0.5% normal heron serum.
  • RIA buffer 50 mM sodium phosphate buffer (pH 7.4), 0.5% BSA, 0.5% Triton-X100, 80 mM NaCl, 25 mM EDTA-2Na and 0.05% NaN
  • Antiserum 200 1 was prepared. The anti-rat ghrelin [G11] antiserum and the anti-Pg-saggrelin [13-28] antiserum were used at final dilutions of 1 / 3,000,000 and 1 / 20,000, respectively. After incubation at 4 ° C. for 12 hours, 125 1-labeled ligand 1001 (20,000 cpm) was added and incubated for another 36 hours. Next, 100 1 of anti-Pseudosiagi antibody was added. After incubation at 4 ° C for 24 hours, free and bound tracers were separated by centrifugation at 3,000 rpm for 30 minutes. The radioactivity of the pellet was quantified using a gamma counter (ARC-600, Aloka, Tokyo). All tests were performed in duplicate at 4 ° C.
  • Both types of antisera showed complete cross-reactivity with human, mouse and rat darelin (2).
  • the anti-rat ghrelin [1-11] antiserum specifically recognizing the Ser 3 n-Ottanoirirido site of dallelin did not recognize deasyl-type darrellin.
  • the cross-reactivity of N-RIA to n-decanoyldarerin and n-hexanoyldarerin is 20% and 0.3%, respectively (2).
  • the anti-rat ghrelin [13-28] antiserum recognized both the deacylated and all-acylated forms of the darelin peptide equally (2).
  • N-RIA N-terminal fragment of rat darelin
  • C-RIA C-terminal fragment
  • CHO-GHSR62 cells which stably express rat GHS-R (ghrelin receptor), were cultured at 4 ⁇ 10 4 cells / well in a flat-bottom 96-well plate (black) (Corning Costar
  • the stomach collected from either the mouse or rat was washed twice with phosphate buffered saline (PH7.4). After measuring the wet weight of each sample, the whole stomach tissue was finely chopped and boiled for 5 minutes in a 10-fold volume of water to inactivate endogenous lipase. After cooling on ice, the boiled sample was adjusted to 1 M acetic acid-20 mM HC1. Peptides were extracted after homogenization using a Polytron Mixer-1 (PT 6100, Kinematica AG., Littan- Luzern, Switzerland). After centrifugation at 15,000 rpm (12,000 X g) for 15 minutes, the supernatant of the isolated extract was lyophilized and stored at -80 ° C. Lyophilized samples were redissolved in RIA buffer or calcium mobilization assay buffer, respectively, prior to Darrelin RIA or potassium mobilization assay.
  • phosphate buffered saline phosphate buffered saline
  • Plasma samples were prepared as previously described (2). Whole blood samples were immediately transferred to cold polypropylene tubes containing EDTA-2Na (1 mg / ml) and aprotune (1,000 kallikrein inactivator units / ml) and centrifuged at 4 ° C. Immediately after the separation of the plasma, the sample was washed with hydrogen chloride at a final concentration of 0.1 N, and then diluted with an equal volume of physiological saline. Samples were loaded onto Sep-Pak C18 cartridges (Waters, Milford, MA) pre-equilibrated with 0.1% trifluoroacetic acid (TFA) and 0.9% NaCl. Wash the cartridge with 0.9% NaCl and 5% acetonitrile (CH CN) /0.1% TFA, then dissolve in 60% CH CN / 0.1% TFA.
  • TFA trifluoroacetic acid
  • Extracted gastric peptides were collected using Sep-Pak Plus C18 cartridges (Waters, Milford, MA) and analyzed by C18 RP-HPLC (Symmetry 300, 3.9 X 150 mm, Waters) (10-60% CH CN / 0.1% TFA linear gradient, flow rate 1.0 ml / min)
  • n-Heptanoyl ghrelin was purified using the same method as described above for Darrelin purification by anti-rat ghrelin [1-1 l] IgG immunoafitik mouth chromatography (22) (22).
  • FLEX station Molecular Devices, Sunnyvale, CA
  • GHS-R ghrelin receptor
  • CHO-GHSR62 ghrelin receptor
  • mice weighing 20-25g were bred under controlled temperature (21-23 ° C) and under light conditions (light on 0700-1900) with free access to food and water.
  • Glyceryl triheptanoate (Fluka Chemie GmbH, Buchs, Switzerland) was mixed with the standard laboratory feed at a concentration of 5% (w / w).
  • the total consumption of glyceryl triheptanoate-containing diet is approximately 13.5 g / mouse, giving each mouse a total of 675 mg of glyceryl triheptanoate.
  • the stomach was chopped and boiled for 5 minutes in quintuple volume of water to inactivate endogenous proteases.
  • the gastric tissue solution was then adjusted to 1 M acetic acid (AcOH) -20 mM HCl and homogenized with a Polytron mixer.
  • the supernatant of these extracts obtained after centrifugation at 20,000 rpm for 30 minutes was previously equilibrated with 0.1% trifluoroacetic acid (TFA) and a Sep-Pak C18 environmental oral cartridge (Waters, Milford, MA).
  • TFA trifluoroacetic acid
  • the cartridge was filled. After washing with 10% acetonitrile (CH CN) /0.1% TFA, the peptide fraction was
  • the n-heptanoyl-modified darelin was purified with a retention time of 18.4 minutes and the molecular weight was determined by mass spectrometry.
  • the amino acid sequence of the purified peptide was analyzed using a protein sequencer (494, Applied Biosystems, Foster City, CA).
  • Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed using a Voyager DE-Pro spectrometer (Applied Biosystems, Foster City, CA) (25). Mass spectra were recorded in reflection mode at an acceleration voltage of 20 kV. 60% acetonitrile (CH
  • n-hexanoic acid C6
  • n-octanoic acid C8
  • n-lauric acid C12
  • n-palmitic acid C16
  • Gastric peptides were extracted from the stomachs of mice fed water and normal control mice (control) fed standard diet and water. After ingestion, acetyl-modified darelin and total (acyl-modified And desacyl) dalelin concentrations were measured. The acyl-modified darelin was measured by N-RIA, and total darelin was measured by C-RIA. The results are shown in Figure 1.
  • N-RIA is very specific for acyl-modified darelin, and the main form of acyl-dallarelin is n-octanoyldarreline, so the concentration of acyl-modified darelin measured by N-RIA is mainly n- It reflects the Ottatanildarellin population.
  • C represents the ratio of acyl-modified darelin Z total darelin. Data represent mean S.D. of darelin concentration in gastric extracts (from lmg wet weight). Statistical significance was indicated by asterisks. *, p * 0.01; **, p * 0.001 vs. control o
  • mice were fed n-hexanoic acid, n-octanoic acid, n-lauric acid or n-palmitic acid for 14 days, and then the gastric concentrations of acyl-modified darelin and total darelin were fed to normal diet and water. The concentration was compared with that obtained in control mice. Gastric concentrations of acyl-modified darelin were significantly increased in mice fed n-octanoic acid (FIG. 1A).
  • n-hexanoic acid n-decanoic acid or n-palmitic acid.
  • exogenous replenishment n-Octanoic acid increased the gastric concentration of n-otatanyl dallerin without increasing the total (acyl-modified and de-acyled) darelin peptides.
  • Ingested triacylglycerol is hydrolyzed in the lumen and is absorbed through the gastrointestinal mucosa as free fatty acids or monoglycerides.
  • ingested triacyldaricerol can function as a source of free fatty acids (26).
  • the mice were given 5% (w / w) glyceryl trihexanoate (C6), glyceryl trioctanoate (C8), and tridecane.
  • the diet was mixed with glyceryl acid (C10) or glyceryl tripalmitate (C16). Two weeks later, gastric peptides were extracted.
  • A represents the concentration of acyl-modified darelin measured by darelin N-RIA.
  • B represents the total darelin concentration measured by darelin C-RIA.
  • C represents the ratio of the concentration of acyl-modified darelin Z total ghrelin.
  • Statistical significance is indicated by an asterisk. *, p ⁇ 0.05; **, p ⁇ 0.01 vs control.
  • Figure 2 shows that glyceryl trioctanoate intake stimulates the production of acyl-modified darelin in gastric tissue (Figure 2A).
  • Figure 2A shows that ingestion of glyceryl trihexanate slightly suppressed the production of acyl-modified dallelin.
  • Mice fed dariseryl trihexanoate with increasing force showed increased levels of n-hexanoyldarellin (FIG. 2A, Table 1).
  • Ingestion of glyceryl tridecanoate and glyceryl tripalmitate had no effect on the production of acyl-modified darellin (FIG. 2A).
  • glyceryl trihexanoate was used to determine the molecular form of the darelin peptide.
  • peaks &, d, h, and k correspond to desacyl darelin
  • peaks b, f, i, and 1 are n-otatanyl (C8: 0) corresponds to ghrelin
  • peaks c, g, j and m correspond to n-decenoyl (C10: l) ghrelin.
  • n-Hexanoyldarellin was extremely low in the stomach of mice fed a normal diet and was not detected by force.
  • glyceryl trihexanoate was given to the mice, the gastric concentration of n-hexanoyldarerin increased dramatically (peak. In these mice, the measured values in control mice (peak b in Figure 3 and Table 1). ), A significant decrease in the concentration of n-otanoyldarellin was also detected (peak 1 in Figure 3 and Table 1). It also increased after ingestion (data not shown).
  • n-decanoyldarellin When glyceryl tridecanoate was given to mice, the gastric concentration of n-decanoyldarellin increased (peak n).
  • the Darrelin peak eluting at the same retention time as synthetic n-butanoyl (C4: 0) ghrelin, n-dodecanoyl (C12: 0) ghrelin and n-palmitoyl (C16: 0) darrelin is glyceryl tributyrate. No power was observed in gastric extracts of mice fed glyceryl trilaurate or glyceryl tripalmitate (data not shown). These data indicate that neither glyceryl tributyrate or glyceryl tripalmitate was converted to dallerin in mice.
  • mice Male C57BL / 6J mice received 5% (w / w) glyceryl trihexanoate (C6: 0-MCT), glyceryl trioctanoate (C8: 0-MCT) or glyceryl tridecanoate (C10: 0-MCT). The mixed diet was given for 14 days.
  • concentrations of l-ghrelin) and n-decanol ghrelin were measured by Darrelin C-RIA after HPLC fractionation. Data represent mean SD from quadruplicate samples.
  • mice fed a 12-hour fasted glyceryl trioctanoate diet 5% w / w.
  • the gastric concentrations of the acyl-modified darelin and total darelin after a certain period of time were measured.
  • Fig. 4 shows the results.
  • A represents the content of an acyl-modified darrellin measured by darrellin N-RIA
  • B represents the total darrellin content measured by darrellin C-RIA.
  • mouse gastric mRNA was quantified by Northern blot analysis 4 days after ingestion of a diet containing glyceryl trioctanoate.
  • Fig. 5 shows the results.
  • Each lane contains 2 ⁇ g of total RNA.
  • the lower panel shows 28S and 18S ribosomal RNA internal controls.
  • FIG. 5 shows that the expression level of gastric darelin mRNA did not change after ingestion of glyceryl trioctanoate.
  • glyceryl trioctanoate increased the gastric content of n-otatanyldalerelin without changing the total darelin concentration, ingestion of glyceryl trioctanoate stimulated only the otatanyl modification step in dalerin peptide synthesis. That is,
  • mice were fed medium-chain triglycerides (MCT), which are not present in natural dietary sources and are not synthesized in mammals. Since n-heptanoic acid (C7: 0), which is a hydrolyzed form of glyceryl triheptanoate, does not naturally exist in mammals, glyceryl triheptanoate was selected as a non-natural free fatty acid source. In addition, it seems that n-heptanoyldarerelin is easily separated from natural darelin by HPLC. Fig. 6 shows the results.
  • peaks a and c corresponding to the retention time of the isolated darelin peptide correspond to de-acyl-type darelin and n-otatanyldarreline, respectively.
  • Fig. 6 Extrapyretic darrellin immunoreactivity was observed only in mice fed dalyseryl triheptanoate, and other free fatty acids or triglycerides tested (n-hexanoic acid, n-octanoic acid, n-lauric acid, n_palmitic acid). Or the corresponding triglyceride form).
  • the retention time of peak b was between n-hexanoyldarerin and n-otatanyldarerin.
  • the stomach tissue parasyl modified darellin of a mouse fed a glyceryl triheptanoate-containing diet for 4 days was purified.
  • the sample from which the anti-rat ghrelin immobility column was also eluted was subjected to HPLC.
  • Fig. 7 shows the results.
  • Peak a was observed only in samples derived from mice treated with glyceryl triheptanoate. Based on HPLC retention time and MALDI-TOF-MS analysis, peak b corresponded to n-otatanyldarellin.
  • the arrows indicate the elution positions of n-hexanoyl (1), n-otatanyl ( ⁇ ) and n-decanoyl (III) dallelin, respectively.
  • peak b in Fig. 7 was identified as n-otatanyl darelin based on the retention time in HPLC.
  • Another peak eluting at a retention time of 18.4 minutes was only observed after glyceryl triheptanoate ingestion.
  • This peak eluted with a retention time between n-hexanoyldarerin and n-otatanyldarerin.
  • the peptide at peak a was purified and subjected to amino acid sequencing analysis and mass spectrometry.
  • the purified peptide obtained from HPLC peak a (Fig. 7) consisted of 28 amino acids and was identical to the amino acid sequence of mouse ghrelin. Matritus-assisted laser desorption ionization time-of-flight mass spectrometry of dallelin-like peptide purified from peak a in Figure 7 was performed today. The results are shown in FIG. 8A.
  • B represents the structure of n-heptanoyl (C7: 0) ghrelin.
  • the estimated molecular weight of the peptide calculated from the m / z value of MALDI-TOF-MS was 3300.9.
  • Modification of the ghrelin n-heptanol group at the Ser 3 residue results in a molecular weight of about 3300.86 in theory (FIG. 8B). This is almost the same as the molecular weight measured by MALDI-TOF-MS. Therefore, it was concluded that the purified peptide at peak a was n-heptanoyldarellin. In the final purification step, No peak is observed. This indicates that the ingested glyceryl triheptanoate can also directly transfer the hydrolyzed n-heptanoyl group to the Ser 3 residue of dallelin.
  • mice fed a diet containing glyceryl triheptanoate for 4 days were tested.
  • the molecular morphology of plasma-derived acyl-modified darelin was determined. That is, plasma samples collected from a control mouse (A) and a glyceryl triheptanoate-treated mouse (B) fed a standard diet were fractionated by HPLC, and ghrelin immunoreactivity was measured by C-RIA. The results are shown in FIG.
  • the arrows indicate the elution positions of desacyl-type darelin (I) and n-otatanyldarrelin ( ⁇ ).
  • the plasma dalelin immunoreactivity was represented by a bar graph.
  • Plasma ghrelin immunoreactivity in control mice was separated into two main peaks (peaks a and b in Fig. 9A) and one small peak (peak c in Fig. 9A).
  • Plasma darellin immunoreactivity in glyceryl triheptanoate-treated mice was separated into two main peaks (peaks d and e in FIG. 9B) and two minor peaks (peaks 1 and g in FIG. 9B).
  • peaks b and e correspond to de-otanoyldarellin
  • peaks c and g correspond to n-otatanyldarerin.
  • the newly appearing peak f showed the same retention time as n-heptanoyldarellin observed in the mouse stomach after glyceryl tryptanoate treatment.
  • Peaks a and d are believed to be the C-terminal portion of the darelin peptide generated by protease digestion, but the exact molecular form has not yet been determined.
  • n-Heptanoyldarellin induces an increase in [Ca2 + ] in GHS-R-expressing cells, and the time course of these [Ca2 + ] i changes is Similar to the changes induced by (Fig. 10).
  • the agonist activity of n-heptanyl ghrelin for GHS-R calculated from the area under the curve (AUC) of the response curve, is about 60% of that of n-otatanyl ghrelin. It is three times higher than that of nildalelin ( Figure 10). Therefore, n-heptanoyldarerin has GHS-R stimulating activity.
  • the present invention opens the way to the molecular mechanism of the modification of darelin to acyl and the identification of the enzyme responsible for the modification.
  • the experimental results suggest that darellin ser Q-acyltransferase, which functions in mice, can catalyze the cascade modification of n-hexanoyl, n-heptanyl, n-otatanyl and n-decanoyl darellin.
  • This type of enzyme did not catalyze the acetyl modification of dallelin after ingestion of glyceryl tripalmitate, long-chain triacylglyceride (LCT) and glyceryl tributyrate, short-chain triacylglyceride (SCT).
  • MCT MCT
  • the present invention provides a method for exogenous or metabolically produced MCFs. Some have been converted to medium-chain acetyl-CoA, suggesting that it may be used for the modification of darellin to acyl.
  • acyltransferases have been identified in mammals.
  • the only enzyme reported to use MCFA as a substrate is cal-tin otatanyl transferase (29, 30), which functions in the j8-oxidation of fatty acids.
  • a member of the serine acyltransferase family has been identified that transfers an acyl group to a serine residue in a target molecule. It includes two serine palmitoyltransferases (31) that function in the biosynthesis of sphingolipids in mammals (31), and one plant serine 0-acetyltransferase gene family in Arabidopsis thaliana (32, 33). included.
  • Protein lipid acyltransferase has also been purified from rat gastric mucosa (34, 35). This enzyme is an endogenous rough microsomal protein that catalyzes the transfer of acyl-CoA to mucosal proteins. Putative darelin ser Q-acyltransferases may have structural similarity to these acyltransferases
  • Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature
  • Matsukura S, Kangawa K, Nakazato M. Ghrelin a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the

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Abstract

Un régulateur pour réguler les fonctions physiologiques de la ghrelin (par exemple, un effet d’augmentation de la concentration intracellulaire en ions calcium, un effet d’activation de la sécrétion de l’hormone de croissance, un effet d’activation de l’ingestion de nourriture, un effet relatif à la régulation de l’accumulation de gras, un effet d’amélioration des fonctions cardiaques ou un effet de stimulation de la sécrétion d’acide gastrique) qui contient un acide gras ayant de 2 à 35 atomes de carbone ou son dérivé.
PCT/JP2004/015413 2004-06-09 2004-10-19 Régulateur pour les fonctions physiologiques de la ghrelin WO2005120484A1 (fr)

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WO2016050754A1 (fr) * 2014-09-30 2016-04-07 Nestec S.A. Composition nutritionnelle à faible teneur en acides gras à chaîne moyenne dans des proportions spécifiques, et ses utilisations
US9561206B2 (en) * 2015-01-07 2017-02-07 The United States Of America, As Represented By The Secretary Of The Navy Use of heptadecanoic acid (C17:0) to detect risk of and treat hyperferritinemia and metabolic syndrome
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CA3060243A1 (fr) 2017-04-17 2018-10-25 The University Of Chicago Matieres polymeres pour l'administration d'acides gras a chaine courte a l'intestin pour des applications de sante humaine et de traitement de maladie
EP3661600A4 (fr) 2017-10-23 2021-08-11 Epitracker, Inc. Analogues d'acides gras et leur utilisation dans le traitement des états liés au syndrome métabolique
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JP5144929B2 (ja) 2013-02-13
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CA2569678C (fr) 2014-01-14
US20080293818A1 (en) 2008-11-27
EP1767198A1 (fr) 2007-03-28
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